Compounds that have evolved naturally to inhibit cell growth have proven invaluable in cancer research and therapy. They often exert their effects potently andspecifically - making them useful probes of signaling pathways. Recent examples include the histone deacetylase inhibitor trapoxin, the immunosuppressant rapamycin, and the Sonic Hedgehog signaling inhibitor cyclopamine. The goal of this project is to determine the targets and mode-of-actionfor several newnatural products - ones we believe have similar potential to drive new cell biology research relevant to human cancer. The compounds also represent lead structures for the development of novel therapeutics. A multi-pronged approach combining classical genetics, reverse genetics, chemistry, cell biology, and biochemistry will be used for these purposes. Specifically, we propose to continue genetic studies of a new mechanism of drug resistance uncovered with the anti-mitotiic hemiasterlin. This is relevant in light of the advanced clinical status of HTI-286, a hemiasterlin derivative currently in Phase II human trials. We also propose to identify the target of the natural product psymberin, which was recently reported to display differential cytotoxicity in the NCI Developmental Therapeutics in Vitro Screening Program.We have achieved a practical total synthesis of this natural product, which allows access to synthetic variants for mode-of-action studies. We have shown psymberin is highly toxic to the nematode C elegans-a basis for genetic screens to identify resistant mutants. Palau'amlne is a rare natural product from the ocean having immunosuppressive and antiproliferative activities. Its synthesis is near completion and similar approaches (genetic and biochemical) will be applied to dissect its mode-of- action. Finally, we will pursue the mode-of-action of the cytotoxin halomon, a molecule shown to inhibit the DMA methyltransferase I enzyme. Relevance to public health: A large fraction of FDAapproved anticancer drugs and many more in development are based upon natural product leads. This research will investigate new natural products that operate by unique mechanisms to inhibit human cancer cell growth.